Metabolic engineering Corynebacterium glutamicum for D-chiro-inositol production.

D-chiro-inositol (DCI) is a potential drug for the treatment of type II diabetes and polycystic ovary syndrome. In order to effectively synthesize DCI in Corynebacterium glutamicum, the genes related to inositol catabolism in clusters iol1 and iol2 were knocked out in C. glutamicum SN01 to generate the chassis strain DCI-1. DCI-1 did not grow in and catabolize myo-inositol (MI). Subsequently, different exogenous and endogenous inosose isomerases were expressed in DCI-1 and their conversion ability of DCI from MI were compared. After fermentation, the strain DCI-7 co-expressing inosose isomerase IolI2 and inositol dehydrogenase IolG was identified as the optimal strain. Its DCI titer reached 3.21 g/L in the presence of 20 g/L MI. On this basis, the pH, temperature and MI concentration during whole-cell conversion of DCI by strain DCI-7 were optimized. Finally, the optimal condition that achieved the highest DCI titer of 6.96 g/L were obtained at pH 8.0, 37 °C and addition of 40 g/L MI. To our knowledge, it is the highest DCI titer ever reported.

Establishment of CRISPR-Cpf1-assisted gene editing tool and engineering of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum.

Corynebacterium glutamicum, an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum. To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6%. In the example of PodhA::PilvBNC replacement, pCS plasmid improved the screening efficiency of second-cross-over bacteria from 5% to over 95%. Then this pCS-assisted gene editing system was applied to improve the supply of precursors and reduce the generation of by-products in the production of 4-hydroxyisoleucine (4-HIL). The 4-HIL titer of one edited strain SC01-TD5IM reached 137.0 ± 33.9 mM, while the weakening of lysE by promoter engineering reduced Lys content by 19.0-47.7% and 4-HIL titer by 16.4-64.5%. These editing demonstrates again the efficiency of this novel CRISPR-Cpf1-assisted gene editing tool, suggesting it as a useful tool for improving the genome editing and metabolic engineering in C. glutamicum.

Association of Lower Extremity Vascular Disease, Coronary Artery, and Carotid Artery Atherosclerosis in Patients with Type 2 Diabetes Mellitus.

The motive of this article is to present the case study of patients to investigate the association between the ultrasonographic findings of lower extremity vascular disease (LEAD) and plaque formation. Secondly, to examine the association between the formation of coronary artery and carotid artery atherosclerosis in patients with type 2 diabetes mellitus. 124 patients with type 2 diabetes (64 males and 60 females with the age group 25-78 years) are considered for the research studies who have registered themselves in the Department of Endocrinology and Metabolism from April 2017 to February 2019. All participants have reported their clinical information regarding diabetes, alcohol consumption, smoking status, and medication. The blood samples from subjects are collected for measurement of HbA1c, total cholesterol, triglycerides, HDL-c, and LDL-c levels. Two-dimensional ultrasound has been used to measure the inner diameter, peak flow velocity, blood flow, and spectral width of the femoral artery, pop artery, anterior iliac artery, posterior tibial artery, and dorsal artery and to calculate the artery stenosis degree. Independent factors of atherosclerosis are determined by multivariate logistic regression analysis. The results are evaluated within the control group and it is found that there is no significant impact of gender, age, and body mass index (P > 0.05) on the lower extremity vascular diseases. Those with smoking, alcohol consumption, hypertension, and dyslipidemia have higher positive rate (P < 0.05). The type 2 diabetes mellitus group has higher diastolic blood pressure and lower triglyceride (P < 0.05). Diastolic blood pressure, HbA1C, total cholesterol, HDL-c, and LDL-C are not remarkably dissimilar between the type 2 diabetes mellitus group and the control group (P > 0.05). Compared with the control group, the type 2 diabetes mellitus group has higher frequency of lower extremity vascular diseases in the dorsal artery than in the pop artery (P < 0.05). The blood flow of type 2 diabetes mellitus group is found to be lower than that of the control group, especially in the dorsal artery (P < 0.05). The blood flow velocity of the dorsal artery is accelerated (P < 0.01). Among 117 patients of type 2 diabetes mellitus (94.35%) with a certain degree of injury, there are 72 cases of type I carotid stenosis (58.06%), 30 cases of type II carotid stenosis (24.19%), and 15 cases of type III carotid stenosis (12.10%). Out of 108 subjects in the control group, there are 84 cases of type 0 carotid stenosis (77.78%), 19 cases of type I carotid stenosis (17.59%), 5 cases of type II carotid stenosis (4.63%), and 0 case of type III carotid stenosis (0.00%). Compared with the control group, carotid stenosis is more common in patients with type 2 diabetes mellitus (P < 0.05). Age, smoking, duration of diseases, systolic blood pressure, and degree of carotid stenosis are found to be associated with atherosclerosis. The findings suggest that the color Doppler ultrasonography can give early warning when applied in patients with carotid and lower extremity vascular diseases to delay the incidence of diabetic macroangiopathy and to control the development of cerebral infarction, thus providing an important basis for clinical diagnosis and treatment.

Effects of tripterygium glycosides on restenosis following endovascular treatment.

The mechanism and associated factors of restenosis following intravascular stent implantation remain to be elucidated. The present two­part experimental and clinical study aimed to investigate the effects of tripterygium glycosides on in­stent restenosis subsequent to intra­arterial therapy. Following endovascular stent implantation in rabbit iliac arteries, post­stent outcomes were evaluated in cyclosporine groups, low­dose and high­dose tripterygium glycosides groups and controls. Post­operative angiography indicated that vessel diameters were similar between groups; however, at 28 days after receiving the therapeutic agents, vessels of the cyclosporine and tripterygium glycosides groups were significantly larger than those of the controls. Furthermore, three groups of patients had comparable baseline levels of interleukin (IL)­10, IL­18 and C­reactive protein, and intima­media thickness. However, 1 month after stent implantation, levels of IL­10 and IL­18 were markedly reduced in the high­ and low­dose tripterygium glycosides groups compared with controls. At 6 months after surgery, the stent patency rate in patients with bare stents was significantly lower than in patients receiving tripterygium glycosides (P≤0.009). In addition, the ankle­brachial index was also higher than in those without tripterygium glycosides (P<0.001). Results of the experimental and clinical studies suggest that tripterygium glycosides may inhibit and possibly aid in the prevention of in­stent restenosis formation following endovascular treatment of lower­extremity artery disease.

Prevention of pulmonary embolisms associated with vena cava filter implantation.

OBJECTIVE: To investigate the prevention of pulmonary embolism (PE) by the implantation of inferior vena cava filters (IVCFs). METHODS: Data of 1058 patients treated for lower extremity deep vein thrombosis (DVT) between January 2005 and January 2012 were analyzed retrospectively and divided into those with and without PE. RESULTS: Of the 1058 patients, 34 had PEs (3.2%) and 1024 did not. Multivariable analyses showed that PEs were less likely to occur in patients with IVCFs than in those without IVCFs (odd ratio [OR] = 0.028, P < 0.001), and were more likely to occur in patients with vena cava thrombosis than in those without vena cava thrombosis (OR = 19.094, P < 0.001). Analyses stratified by DVT site showed that vena cava thrombosis was the only risk factor of PE for patients with a left DVT (crude OR = 12.814, P < 0.001), and IVCF implantation was the only protective factor for PE for patients with a right or bilateral DVT (crude OR = 0.028, P = 0.001). IVCF patency rate for follow-up was 98.7% with no filter migration, declination, or failure of expansion. CONCLUSIONS: IVCFs can reduce the occurrence of PEs in patients with DVTs.

[Leihong granule intervened in-stent restenosis after endovascular therapy for lower extremity arterial occlusive diseases: a clinical observation].

OBJECTIVE: To observe the intervention effect of Leihong Granule (LG) in in-stent restenosis (ISR) after endovascular therapy for lower extremity arterial occlusive diseases (LEAOD). METHODS: Recruited 80 LEAOD patients who successfully underwent endovascular therapy (balloon dilation and stent implantation) were randomly assigned to two groups, the control group and the LG group, 40 in each group. Patients in the control group received basic treatment, while those in the LG group additionally took LG for 3 months. Plasma levels of IL-10, IL-18, CRP, and the intima-media thickness (IMT) of lower extremity artery were observed in the two groups between and after treatment. The rate of stent patency, ABI, intermittent claudication, rest pain, and the incidence of amputation the two groups were recorded and observed in the two groups. RESULTS: In the control group, serum levels of IL-10, IL-18, CRP, and IMT were significantly higher one month after surgery than before surgery (P < 0.05). There was no significant difference in serum levels of IL-10, IL-18, CRP, or IMT between the two groups before surgery (P > 0.05). These indices were obviously lower in the LG group than in the control group after surgery (P < 0.05). Compared with the control group, the incidence rates of intermittent claudication and the rest pain at 6 months and 12 months after surgery significantly decreased (P < 0.05). The stent patency rate at 6 months and 12 months after surgery, and ABI were significantly higher than those of the control group (P < 0.05). There was no statistical difference in the amputation rate between the two groups (P > 0.05). CONCLUSION: LG might effectively improve ischemic symptoms of affected limbs possibly through lowering the ISR rate after endovascular therapy for LEAOD through preventing immunosuppressive actions.